Molecular mechanisms of TRPV5 gating

项目总结/摘要 肾结石，通常被称为肾结石，是一种常见的疾病，影响约30 数百万美国人肾结石形成的最关键的危险因素之一是高钙尿症，或高钙尿症。 尿钙（Ca 2+）。瞬时受体电位香草酸5型（TRPV 5）通道， 主要在肾脏中表达，已经发现对于Ca 2+重吸收到血液中是必需的。损失 TRPV 5的功能障碍或功能障碍已被证明会严重增加尿Ca 2+水平和肾功能衰竭的发生。 石头TRPV 5存在于肾单位上皮细胞的顶膜中，并允许Ca 2+从 尿液沿着其浓度梯度。在不存在调节剂的情况下，已经提出TRPV 5是 组成活跃。内源性调节剂如钙调蛋白（CaM）和磷脂酰肌醇4，5- 已经发现二磷酸（PI（4，5）P2）分别稳定TRPV 5处于闭合或开放构象。 蛋白激酶C（PKC）介导的TRPV 5在Ser 299和Ser 654残基的磷酸化已被证明是 提高了信道的开通概率。小分子抗真菌药，如益康唑和咪康唑， 证明抑制TRPV 5。然而，这种通道调节和门控的分子细节很差， 明白因此，本提案的目标是结合使用低温电子显微镜（cryo-EM） 生物化学，电生理学和计算方法，以揭示分子机制， TRPV 5门控。在原子水平上对TRPV 5的深入研究将为靶向药物铺平道路 用于控制和治疗高钙尿症和肾结石的新发现。

PROJECT SUMMARY/ABSTRACT Nephrolithiasis, generally known as kidney stones, is a common disease that affects approximately 30 million Americans. One of the most critical risk factors for kidney stone formation is hypercalciuria, or high levels of urine calcium (Ca2+). The transient receptor potential vanilloid type 5 (TRPV5) channel, which is primarily expressed in the kidney, has been found to be essential for reabsorption of Ca2+ into the blood. Loss or dysfunction of TRPV5 has been shown to severely increase urine Ca2+ levels and the occurrence of kidney stones. TRPV5 is found in the apical membrane of the nephron epithelium and allows Ca2+ reabsorption from the urine along its concentration gradient. In the absence of modulators, TRPV5 has been proposed to be constitutively active. Endogenous modulators such as calmodulin (CaM) and phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) have been found to stabilize TRPV5 in the closed or open conformation, respectively. Protein kinase C (PKC)-mediated TRPV5 phosphorylation at Ser299 and Ser654 residues has been shown to enhance open probability of the channel. Small molecule antifungals like econazole and miconazole have been demonstrated to inhibit TRPV5. However, molecular details of this channel modulation and gating are poorly understood. Therefore, the goal of this proposal is to utilize cryo-electron microscopy (cryo-EM) in conjunction with biochemical, electrophysiological and computational approaches to uncover the molecular mechanisms of TRPV5 gating. An in-depth investigation of TRPV5 at the atomic level will pave the way for targeted drug discovery for the control and treatment of hypercalciuria and nephrolithiasis.